Cellular and metabolic specificity in the interaction of adhesion proteins with collagen and with cells

Fibronectin mediates the adhesion of fibroblasts to collagen substrates, binding first to the collagen and then to the cells. We report here that the interaction of the cells with the fibronectin-collagen complex is blocked by specific gangliosides, GD_{1 a} and GT₁, and that the sugar moieties of these gangliosides contain the inhibitory activity. The gangliosides act by binding to fibronectin, suggesting that they may be the cell surface receptor for fibronectin. Evidence is presented that other adhesion proteins or mechanisms of attachment exist for chondrocytes, epidermal cells, and transformed tumorigenic cells, since adhesion of these cells is not stimulated by fibronectin. Chondrocytes adhere via a serum factor that is more temperature-sensitive and less basic than fibronectin. Unlike that of fibroblasts chondrocyte adhesion is stimulated by low levels of gangliosides. Epidermal cells adhere preferentially to type IV (basement membrane) collagen but at a much slower rate than fibroblasts or chondrocytes. This suggests that these epidermal cells synthesize their own specific adhesion factor. Metastatic cells cultured from the T241 fibrosarcoma adhere rapidly to type IV collagen in the absence of fibronectin and do not synthesize significant amounts of collagen or fibronectin. Their growth, in contrast to that of normal fibroblasts, is unaffected by a specific inhibitor of collagen synthesis. These data indicate the importance of specific collagens and adhesion proteins in the adhesion of certain cells and suggest that a reduction in the synthesis of collagen and of fibronectin is related to some of the abnormalities observed in transformed cells.     

Neutrophils, monocytes, and lymphocytes bind to cytokine-activated kidney glomerular endothelial cells through L-selectin (LAM-1) in vitro.

The role of L-selectin (LAM-1) as a regulator of leukocyte adhesion to kidney microvascular glomerular endothelial cells was assessed in vitro by using L-selectin-directed mAb and an L-selectin cDNA-transfected cell line. The initial attachment of neutrophils, monocytes, and lymphocytes to TNF-activated bovine glomerular endothelial cells was significantly inhibited by the anti-LAM1-3 mAb. Under static conditions, anti-LAM1-3 mAb inhibited neutrophil adhesion by 15 +/- 5%, whereas the anti-LAM1-10 mAb, directed against a functionally silent epitope of L-selectin, was without effect. The binding of a CD18 mAb inhibited adhesion by 47 +/- 6%. In contrast, when the assays were carried out under nonstatic conditions or at 4 degrees C, the anti-LAM1-3 mAb generated significantly greater inhibition (approximately 60%). CD18-dependent adhesion was minimal (approximately 10%) under these conditions. TNF-activated glomerular endothelial cells also supported adhesion of a mouse pre-B cell line transfected with L-selectin cDNA, but not wild-type cells. This process was also inhibited by the anti-LAM1-3 mAb. Leukocyte adhesion to unstimulated endothelial cells was independent of L-selectin, but, after TNF stimulation, L-selectin-mediated adhesion was observed at 4 h, with maximal induction persisting for 24 to 48 h. Leukocyte adhesion was not observed if glomerular endothelial cells were exposed to TNF in the presence of RNA or protein synthesis inhibitors. Leukocyte attachment to TNF-activated glomerular endothelial cells was also partially inhibited by treatment of the cells with mannose-6-phosphate or phosphomannan monoester, a soluble complex carbohydrate, or by prior treatment of glomerular endothelial cells with neuraminidase, suggesting that the glomerular endothelial cell ligand shares functional characteristics with those expressed by lymph node and large vessel endothelial cells. These data suggest that TNF activation induced the biosynthesis and surface expression of a ligand(s) for L-selectin on glomerular endothelial cells, which supports neutrophil, monocyte, and lymphocyte attachment under nonstatic conditions.     

Immobilized gellan sulfate surface for cell adhesion and multiplication: development of cell-hybrid biomaterials using self-produced fibronectin

A new concept for cell-hybrid biomaterial is proposed in which human unbilical vein endothelial cells (HUVEC) are adhered to an immobilized gellan sulfate (GS) surface. Extra domain A containing fibronectin (EDA(+)FN) released from HUVEC is necessary for cell adhesion and multiplication. The material design in this study is based on these self-released cell adhesion proteins. The interaction between GS and EDA(+)FN was evaluated using the affinity constant (KA); the value obtained was 1.03x10(8) (M(-1)). These results suggest that the adhesion of HUVEC to GS may be supported by the adhesion of EDA(+)FN to GS. We also found that this new material adheres to HUVEC, allowing the reintroduction of EDA(+)FN, which is self-produced by the cell. This material is relatively easy to produce, not requiring the usual coating of adhesion proteins in pretreatment.     

Fibronectin–collagen binding and requirement during cellular adhesion

Fibronectin isolated from human plasma and from the extracellular matrices of cell monolayers mediates the attachment in vitro and spreading of trypsin-treated cells on a collagen substratum. Fibronectin-dependent kinetics of cellular attachment to collagen were studied for several adherent cell types. It was shown that trypsin-treated human umbilical-cord cells, mouse sarcoma CMT81 cells, endothelial cells, and human fibroblasts from a patient with Glanzmann's disease were completely dependent on fibronectin for their attachment to collagen, whereas guinea-pig and monkey smooth-muscle cells and chick-embryo secondary fibroblasts displayed varying degrees of dependence on fibronectin for their attachment. Radiolabelled human plasma fibronectin possessed similar affinity for collagen types I, II and III from a variety of sources. The fibronectin bound equally well to the collagens with or without prior urea treatment. However, in the fibronectin-mediated adhesion assay using PyBHK fibroblasts, a greater number of cells adhered and more spreading was observed on urea-treated collagen. Fibronectin extracted from the extracellular matrix of chick-embryo fibroblasts and that purified from human plasma demonstrated very similar kinetics of complexing to collagencoated tissue-culture dishes. Fibronectin from both sources bound to collagen in the presence of 0.05–4.0m-NaCl and over the pH range 2.6–10.6. The binding was inhibited when fibronectin was incubated with 40–80% ethylene glycol, the ionic detergents sodium dodecyl sulphate and deoxycholate, and the non-ionic detergents Nonidet P-40, Tween 80 and Triton X-100, all at a concentration of 0.1%. From these results we proposed that fibronectin–collagen complexing is mainly attributable to hydrophobic interactions.     

Synthesis of lipopolyhydroxylalkyleneamines for gene delivery

Various bis(2-hydroxy-3-chloropropyl)alkylamines were synthesized by coupling primary amine with epichlorohydrin and utilized as a monomer to react with ethylenediamine (EDA), N,N'-dimethylethylenediamine (DMEDA), or tetramethylethylenediamine (TMEDA) to generate a series of lipopolyhydroxylalkyleneamines. The number- and weight-average molecular weight (Mn and Mw) and polydispersity index (Mw/Mn) of the lipopolyhydroxylalkyleneamines were dependent on reactant solvent and reaction temperature. The compounds with EDA as backbone have better transfection activity and lower toxicity than those with DMEDA and TMEDA as backbone.     

Fibronectin-binding protein acts as Staphylococcus aureus invasin via fibronectin bridging to integrin alpha5beta1

The ability of Staphylococcus aureus to invade mammalian cells may explain its capacity to colonize mucosa and to persist in tissues after bacteraemia. To date, the underlying molecular mechanisms of cellular invasion by S. aureus are unknown, despite its high prevalence and difficulties in treatment. Here, we show cellular invasion as a novel function for an S. aureus adhesin, previously implicated solely in attachment. S. aureus , but not S. epidermidis , invaded epithelial 293 cells in a temperature- and F-actin-dependent manner. Formaldehyde-fixed and live bacteria were equally invasive, suggesting that no active bacterial process was involved. All clinical S. aureus isolates analysed, but only a subset of laboratory strains, were invasive. Fibronectin-binding proteins (FnBPs) acted as S. aureus invasins, because: (i) FnBP deletion mutants of invasive laboratory strains lost invasiveness; (ii) expression of FnBPs in non-invasive strains conferred invasiveness; and (iii) the soluble isolated fibronectin-binding domain of FnBP (D1–D4) completely blocked invasion. Integrin α₅β₁ served as host cell receptor, which interacted with staphylococcal FnBPs through cellular or soluble fibronectin. FnBP-deficient mutants lost invasiveness for epithelial cells, endothelial cells and fibroblasts. Thus, fibronectin-dependent bridging between S. aureus FnBPs and host cell integrin α₅β₁ is a conserved mechanism for S. aureus invasion of human cells. This may prove useful in developing new therapeutic and vaccine strategies for S. aureus infections.     

Integrin-like substrate adhesion in RTG-2 cells, a fibroblastic cell line derived from rainbow trout

Fluorometric cell attachment assays together with competitive inhibitors of adhesion were used to probe for the presence of integrins, a diverse family of heterodimeric cell-surface glycoproteins involved in cell-cell and cell-extracellular matrix adhesion, in the fibroblastic rainbow trout cell line, RTG-2. The adhesive properties of this cell line were evaluated. RTG-2 cells adhered poorly to TC plastic in the absence of serum but as little as 2.5% fetal bovine serum allowed over 75% of the cells to attach after 5 h. Surfaces coated with the extracellular matrix proteins collagen I, collagen IV, fibrin, fibrinogen, or fibronectin were able to support attachment of RTG-2 cells. Adhesion of RTG-2 cells to fibronectin varied linearly with fibronectin coating densities in the range 0 to 65 ng/mm(2). Oligopeptides containing the sequence Arg-Gly-Asp (RGD) caused dose-dependent inhibition of adhesion to microtiter plates coated with fibrin, fibrinogen, and fibronectin, whereas attachment to collagen I and collagen IV was less severely affected. In all cases, peptides containing Arg-Gly-Glu (RGE) or Asp-Gly-Arg (DGR) sequences caused no reduction of cell attachment. Since many integrins mediate adhesion by binding to RGD sequences in their target ligands, these results suggest the presence of integrin-like adhesion molecules on the surface of RTG-2 cells.     

Differential effects of laminin, intact type IV collagen, and specific domains of type IV collagen on endothelial cell adhesion and migration

Laminin and type IV collagen were compared for the ability to promote aortic endothelial cell adhesion and directed migration in vitro. Substratum-adsorbed IV promoted aortic endothelial cell adhesion in a concentration dependent fashion attaining a maximum level 141-fold greater than controls within 30 min. Aortic endothelial cell adhesion to type IV collagen was not inhibited by high levels (10(-3) M) of arginyl-glycyl-aspartyl-serine. In contrast, adhesion of aortic endothelial cells on laminin was slower, attaining only 53% of the adhesion observed on type IV collagen by 90 min. Type IV collagen when added to the lower well of a Boyden chamber stimulated the directional migration of aortic endothelial cells in a concentration dependent manner with a maximal response 6.9-fold over control levels, whereas aortic endothelial cells did not migrate in response to laminin at any concentration (.01-2.0 X 10(-7) M). Triple helix-rich fragments of type IV collagen were nearly as active as intact type IV collagen in stimulating both adhesion and migration whereas the carboxy terminal globular domain was less active at promoting adhesion (36% of the adhesion promoted by intact type IV collagen) or migration. Importantly, aortic endothelial cells also migrate to substratum adsorbed gradients of type IV collagen suggesting that the mechanism of migration is haptotactic in nature. These results demonstrate that the aortic endothelial cell adhesion and migration is preferentially promoted by type IV collagen compared with laminin, and has a complex molecular basis which may be important in angiogenesis and large vessel repair.     

Staphylococcus aureus fibronectin binding protein-A induces motile attachment sites and complex actin remodeling in living endothelial cells

Staphylococcus aureus fibronectin binding protein-A (FnBPA) stimulates alpha5beta1-integrin signaling and actin rearrangements in host cells. This eventually leads to invasion of the staphylococci and their targeting to lysosomes. Using live cell imaging, we found that FnBPA-expressing staphylococci induce formation of fibrillar adhesion-like attachment sites and translocate together with them on the surface of human endothelial cells (velocity approximately 50 microm/h). The translocating bacteria recruited cellular actin and Rab5 in a cyclic and alternating manner, suggesting unsuccessful attempts of phagocytosis by the endothelial cells. Translocation, actin recruitment, and eventual invasion of the staphylococci was regulated by the fibrillar adhesion protein tensin. The staphylococci also regularly produced Neural Wiskott-Aldrich syndrome protein-controlled actin comet tails that further propelled them on the cell surface (velocity up to 1000 microm/h). Thus, S. aureus FnBPA produces attachment sites that promote bacterial movements but subvert actin- and Rab5 reorganization during invasion. This may constitute a novel strategy of S. aureus to postpone invasion until its toxins become effective.     

Growth of endothelial and HeLa cells on a new multipurpose microcarrier that is positive,negative or collagen coated

A new cell culture microcarrier that can be covalently bonded by cell attachment proteins and can be thin-sectioned for electron microscopy was synthesized. It was easily made by sulfonating cross-linked polystyrene beads for a negative surface charge followed by covalent attachment of polyethylenimine for a positive charge. Cell attachment proteins, e.g. collagen, was covalently bonded directly to the microcarrier using a carbodiimide or after activating the microcarrier surface with glutaraldehyde. HeLa-S3 cells attached, spread and grew to confluence more efficiently on the positive microcarriers and those coated with collagen than on the negative ones. Endothelial cells grew best on those with a negative surface charge. The nature of the microcarrier surface was not the only aspect involved in cell adhesion but also the type of serum proteins adsorbed. Qualitatively different proteins coated the microcarriers depending upon whether the carrier was negative, positive or coated with collagen. Comparison of various types of available microcarriers indicated that the modified cross-linked polystyrene beads used here were best for transmission and scanning electron microscopy. Endothelial cells grown on the microcarriers had the same ultrastructure as cells grown in monolayers in culture dishes. Of a variety of microcarriers tested the modified cross-linked polystyrene beads were the only ones that could be used for both ultrastructural and biochemical techniques.     

Effects of DNP on the cell surface properties of marine bacteria and its implication for adhesion to surfaces

Abstract

The effect of 2, 4-dinitrophenol (DNP) on the extracelluar polysaccharides (EPS), cell surface charge, and the hydrophobicity of six marine bacterial cultures was studied, and its influence on attachment of these bacteria to glass and polystyrene was evaluated. DNP treatment did not influence cell surface charge and EPS production, but had a significant effect on hydrophobicity of both hydrophilic (p = 0.05) and hydrophobic (p = 0.01) cultures. Significant reduction in the attachment of all the six cultures to glass (p = 0.02) and polystyrene (p = 0.03) was observed after DNP treatment. Moreover, hydrophobicity but not the cell surface charge or EPS production influenced bacterial cell attachment to glass and polystyrene. From this study, it was evident that DNP treatment influenced bacterial cell surface hydrophobicity, which in turn, reduced bacterial adhesion to surfaces.     

Heparin inhibits the attachment and growth of Balb/c-3T3 fibroblasts on collagen substrata.

In investigating the role of cell-extracellular matrix interactions in cell adhesion and growth control, the effects of heparin on cell-collagen interactions were examined. Exponentially growing Balb/c-3T3 fibroblasts were radiolabelled with 3H-thymidine and detached from tissue culture surfaces using EDTA, and cell attachment to various types of collagen substrata was assayed in the presence or absence of heparin or other glycosaminoglycans (GAGs) or dextran sulfate (40 K). Cells attached readily (70-90%) to films of types I and V, but not to type III collagen. The number of cells bound to types I and V collagen films was inhibited by 10-50% when heparin was present from 0.1-100 micrograms/ml. Cell-collagen attachment was also inhibited by dextran sulfate, and to a lesser extent by dermatan sulfate, but chondroitin sulfates A and C and hyaluronic acid showed no effect. Heparin was active even at early time points in the adhesion assay, suggesting it may disrupt cell-collagen attachment. To study the effects of heparin in modulating cell growth on collagen, growth arrested cells cultured on type I collagen films were serum stimulated in the presence of heparin or other GAGs for 3 days. Growth was inhibited (greater than 40%) only by heparin and dextran sulfate. Interaction of heparin fragments (Mr less than or equal to 6KD) with type I collagen was analyzed by affinity co-electrophoresis (Lee and Lander, 1991) and showed higher affinity heparin binding to native as compared with denatured collagen. These data suggest that sites within native collagen may mediate Balb cell-collagen and heparin-collagen interactions, and such interactions may be relevant towards understanding heparin's antiproliferative activity in vivo and in vitro.     

Thrombospondin-induced attachment and spreading of human squamous carcinoma cells

Thrombospondin (TSP) induced the attachment and spreading of human squamous carcinoma cells on plastic culture dishes and dishes coated with type I or type IV collagen. Increased adhesion was detected as early as 15 min after treatment. Dose-response studies indicated that 1-5 micrograms of TSP per 35 mm (diameter) culture dish was sufficient to induce a response and that a half-maximal response occurred at 10 micrograms of TSP/dish. The squamous carcinoma cells synthesized TSP as indicated by biosynthetic labeling experiments. TSP was secreted (or shed) into the culture medium by these cells and also became bound to the cell surface. TSP also promoted adhesion of human keratinocytes, fibroblasts and fibrosarcoma cells but did not induce attachment or spreading of human melanoma or glioma cells, although these cells did respond to laminin.     

Ability of various inserts to promote endothelium cell culture for the establishment of coculture models

To select an insert suitable for human umbilical vein endothelial cell (HUVEC) culture, we compared several available inserts of 0.2 to 0.45 m porosity: Cellagen (ICN), Transwell-COL (Costar), Millicell-HA and CM (Millipore), Anopore (Nunc), Cyclopore (Falcon) in comparison with a control surface (Thermanox). The requirements were: (i) to promote attachment, adhesion and proliferation of HUVEC (judged by [³H]thymidine incorporation into DNA at days 1, 3, 7); (ii) to allow HUVEC visualization by inverted, fluorescence microscopy for uptake of DiI-Ac-LDL and scanning electron microscopy, performed at day 9 after seeding.Because Transwell and Cellagen are collagen precoated and CM has to be coated for cell culture, we performed collagen coating (types I + III or IV) for non-pretreated inserts for the purpose of comparison. Our preferences comprise Transwell-COL, Cyclopore not coated or coated (whatever the collagen type), and Cellagen. However, on a quality/price ratio criterion, Cyclopore, even uncoated, is the insert of choice. The HA, CM and Anopore inserts, even coated, do not allow HUVEC growth but do not alter positive uptake of acetylated LDL.Abbreviations HUVEC human umbilical vein endothelial cells     

Modification of native collagen with cell‐adhesive peptide to promote RPE cell attachment on Bruch's membrane

Current efforts to reverse loss of visual function due to Age‐related Macular Degeneration point to the restoration of the Retinal Pigment Epithelial (RPE) layer. Restoration of the RPE layer involves replacing lost RPE cells as well as addressing the degeneration of the underlying Bruch's membrane (BM). To advance the potential of using donor BM, we present a strategy to achieve specific and controllable modification of the inner collagenous layer (ICL) of the Bruch's membrane. In particular, interaction between a collagen binding peptide (CBP) sequence with exposed collagen fibers on the ICL surface is utilized to anchor bioactive molecules. Here, a cell‐adhesion sequence is added to the collagen binding sequence to promote attachment and survival of ARPE‐19. First, the binding specificity of the CBP sequence is verified with a fluorescent binding assay. Subsequently, the effect of modification using the peptide is studied qualitatively using confocal fluorescent imaging and quantitatively through a cell proliferation assay. Results of these experiments indicate that the peptide sequence binds specifically to collagen fibers. Additionally, modification using the peptide enhanced cell adhesion, allowing large uniform cell networks to be formed on the surface. Furthermore, modification with the peptide also delayed the onset of apoptosis on adherent cells. Biotechnol. Bioeng. 2009;102: 1723–1729. © 2008 Wiley Periodicals, Inc.     

Cationic polyelectrolyte hydrogel fosters fibroblast spreading, proliferation, and extracellular matrix production: Implications for tissue engineering

Fibrous encapsulation is known to occur to many prosthetic implants and is thought to be due to the cells not adhering adequately to the surface. For developing new materials able to enhance cellular adhesion by mimicking extracellular matrix components, polyelectrolyte polymers, characterized by tunable surface charges, have been proposed. Here we demonstrate that panoply of cell functions over a two-dimensional substratum is influenced by surface charge. We have at first generated structurally related polyelectrolyte substrata varying in their positive surface charge amount and subsequently evaluated a variety of behaviors of human primary fibroblasts seeded on these polymers. The proportion of adherent, spreading, and proliferating cells was increased significantly on cationic hydrophilic surfaces when compared with the neutral base surface. The extent of cell spreading correlated with cytoskeleton organization as assessed using immunofluorescence techniques. In the key experiment, the presence of cationic charges on cell adhesion-resistant neutral surface increased the synthesis of collagen I and III, the release of their metabolites, and the expression of their mRNA by fibroblasts. Interestingly, the scarce collagen deposits on neutral polymer consisted, for the most part, of collagen I while collagen III was present only in traces probably due to the secretion of metalloproteinase-2 by non-adherent fibroblasts. Taken together, these results show that polyelectrolyte films may promote the attachment of fibroblast cells as well as their normal secretory phenotype. Both effects could be potentially useful in integrating soft connective tissue to the implant, decreasing the chance of its fibrous encapsulation.     

Anthrax toxin receptor 1/tumor endothelium marker 8 mediates cell spreading by coupling extracellular ligands to the actin cytoskeleton

Tumor endothelial marker 8 (TEM8) is induced in tumor-associated vasculature and acts as a receptor for Protective Antigen (PA), the cell-binding component of the anthrax toxin determinant for toxin entrance into cells. However, the normal function for TEM8 remains unknown. We show that TEM8 functions as an adhesion molecule mediating cell spreading on immobilized PA and collagen I. The mechanism for TEM8 interaction with collagen I was cell type-specific, because binding to collagen I was abrogated by beta1 integrin function blocking antibody in HEK293 cells, but not in primary synovial rabbit fibroblasts. Binding to PA remained unaffected by the addition of beta1 integrin function blocking antibody. Whereas the extracellular and transmembrane domains of TEM8 were sufficient to provide cell attachment, the intracellular domain was critical for spreading. Fusion of the cytosolic domain of TEM8 to the IL-2 receptor, conferred cell-spreading capability on IL-2 receptor antibody substrates. The cytoplasmic domain mediated linkage with the actin cytoskeleton as it co-precipitated actin and determined partitioning of TEM8 to the actin-containing detergent insoluble cellular fraction. TEM8 anchorage to actin was relevant as spreading was inhibited by the cytoskeleton-disrupting drug cytochalasin D, but persisted in the presence of the microtubule-depolymerizing drug nocodazole, and in cells lacking intermediate filaments. Thus, our results indicate that TEM8 is a new adhesion molecule linking collagen I or PA to the actin cytoskeleton.     

Cation Type Specific Cell Remodeling Regulates Attachment Strength

Single-molecule experiments indicate that integrin affinity is cation-type-dependent, but in spread cells integrins are engaged in complex focal adhesions (FAs), which can also regulate affinity. To better understand cation-type-dependent adhesion in fully spread cells, we investigated attachment strength by application of external shear. While cell attachment strength is indeed modulated by cations, the regulation of integrin-mediated adhesion is also exceedingly complex, cell specific, and niche dependent. In the presence of magnesium only, fibroblasts and fibrosarcoma cells remodel their cytoskeleton to align in the direction of applied shear in an α₅-integrin/fibronectin-dependent manner, which allows them to withstand higher shear. In the presence of calcium or on collagen in modest shear, fibroblasts undergo piecewise detachment but fibrosarcoma cells exhibit increased attachment strength. These data augment the current understanding of force-mediated detachment by suggesting a dynamic interplay in situ between cell adhesion and integrins depending on local niche cation conditions.     

Rapid communication Improved growth of cultured brain microvascular endothelial cells on glass coated with a biological matrix

An improved method for culturing primary rat brain capillary endothelial cells on glass has been developed, using a corneal extracellular matrix coat. Since the collagen-coated plastic attachment surface conventionally used for primary cultures of brain microvascular endothelium gives a high level of background fluorescence in microfluorimetric studies, an alternative attachment surface was tested involving no plastic element. Five substrata combinations were examined and a new combination of glass and corneal endothelial extracellular matrix coat was found to provide excellent cell adhesion, culture growth and purity. Other established substrata combinations tested for comparison, either involved plastic, or used glass with collagen or carbodiimide and collagen coating but the last two gave poor endothelial cell adhesion and growth. Our method using this new attachment surface combination results in stable and pure endothelial cultures, as verified by immunocytochemistry, which are suitable for fluorimetric investigations.     

Improved growth of cultured brain microvascular endothelial cells on glass coated with a biological matrix

An improved method for culturing primary rat brain capillary endothelial cells on glass has been developed, using a corneal extracellular matrix coat. Since the collagen-coated plastic attachment surface conventionally used for primary cultures of brain microvascular endothelium gives a high level of background fluorescence in microfluorimetric studies, an alternative attachment surface was tested involving no plastic element. Five substrata combinations were examined and a new combination of glass and corneal endothelial extracellular matrix coat was found to provide excellent cell adhesion, culture growth and purity. Other established substrata combinations tested for comparison, either involved plastic, or used glass with collagen or carbodiimide and collagen coating but the last two gave poor endothelial cell adhesion and growth. Our method using this new attachment surface combination results in stable and pure endothelial cultures, as verified by immunocytochemistry, which are suitable for fluorimetric investigations.